Tyrosine kinase inhibitors have been some of the most successful cancer treatments of the last 10 years. However, dose limiting toxicities and the rapid emergence of resistance suggest that significant improvements can be made to the compounds entering the clinic. It is clear that off-target activity can result in cardiovascular defects, immune system suppression, and liver toxicity. One way to avoid these problems is to generate highly specific inhibitors that avoid off-target side effects. However, target promiscuity is in many cases responsible for clinical efficacy, with inhibition of multiple redundant or compensatory kinases necessary for clinical effect. Thus, a major challenge facing kinase drug discovery today is how to optimize second generation inhibitors to block selected kinases, while avoiding broad spectrum activity and the associated toxicities. Unfortunately, platforms that would enable the efficient selection of multi-targeted inhibitors are currently not available as th focus has routinely been on the development of potent and selective inhibitors to a single kinase. Although kinome-wide panels of in vitro biochemical assays are available for selectivity measurements, these assays are cost and time prohibitive to perform on a large scale early in drug discovery and do not adequately replicate the complex cellular environment of the kinase. In our Phase I application we developed a novel adaptation of the existing BaF3 assay platform to measure signaling directly downstream of the transformed kinase, dramatically reducing the false positives normally observed with kinase inhibitor screening. This improvement is a fundamental leap forward in tyrosine kinase cell-based assays that allows this platform to be used with even the most promiscuous inhibitors and at the earliest phases of drug development, including secondary screening and lead identification. In order to enable compound optimization against multiple kinases, the new assay was multiplexed using Primity's CellCode technology such that nine kinase-transformed cell lines could be analyzed in a single well of a 384 well plate. A screen of 300 kinase inhibitors revealed the power of being able to screen for target activity and selectivity simultaneously. In this Phase II application, we propose the extension of this platform to all 90 known members of the tyrosine kinome. The successful completion of these studies will create the first comprehensive, cell-based kinase assay platform for selectivity measurements, probe development, and the selection of next generation multi- targeted inhibitors.